Non-competitive resource exploitation within mosquito shapes within-host malaria infectivity and virulence.

Malaria is a fatal human parasitic disease transmitted by a mosquito vector. Although the evolution of within-host malaria virulence has been the focus of many theoretical and empirical studies, the vector's contribution to this process is not well understood. Here, we explore how within-vector resource exploitation would impact the evolution of within-host Plasmodium virulence. By combining within-vector dynamics and malaria epidemiology, we develop a mathematical model, which predicts that non-competitive parasitic resource exploitation within-vector restricts within-host parasite virulence. To validate our model, we experimentally manipulate mosquito lipid trafficking and gauge within-vector parasite development and within-host infectivity and virulence. We find that mosquito-derived lipids determine within-host parasite virulence by shaping development (quantity) and metabolic activity (quality) of transmissible sporozoites. Our findings uncover the potential impact of within-vector environment and vector control strategies on the evolution of malaria virulence.

The role of microRNAs in Anopheles biology-an emerging research field.

In the last years, microRNAs (miRNAs) have been established as important post-transcriptional regulators of critical physiological processes in animals and plants. Here, we summarize what is known about miRNA biosynthesis, expression and function in the malaria vector mosquito Anopheles gambiae with a particular emphasis on the mosquito-parasite interactions. We discuss the important gaps in the current knowledge, including the potential of miRNA manipulation for future vector control strategies.

Mosquito midguts and malaria: cell biology, compartmentalization and immunology.

The malaria parasite Plasmodium has an absolute requirement for both a vertebrate and a mosquito host in order to complete its life cycle, and its interactions with the latter provide the focus for this review. The mosquito midgut represents one of the most challenging environments for the survival and development of Plasmodium, and is thus also one of the most attractive sites for novel targeted malaria control strategies. During their attempts to cross the midgut epithelium en route to the salivary glands, motile ookinetes are swiftly detected and labelled by mosquito recognition factors and targeted for destruction by a variety of immune responses that recruit killing factors both from the midgut and from other tissues in the surrounding body cavity. The exact interplay between these factors and the parasite is highly species- and strain-specific, as are the timing and the route of parasite invasion. These features are paramount to determining the success of the infection and the vector competence of the mosquito. Here we discuss recent advances in genomic analyses, coupled with detailed microscopical investigations, which are helping to unravel the identity and roles of the major players of these complex systems.

Conserved role of a complement-like protein in phagocytosis revealed by dsRNA knockout in cultured cells of the mosquito, Anopheles gambiae.

We characterize a novel hemocyte-specific acute phase glycoprotein from the malaria vector, Anopheles gambiae. It shows substantial structural and functional similarities, including the highly conserved thioester motif, to both a central component of mammalian complement system, factor C3, and to a pan-protease inhibitor, alpha2-macroglobulin. Most importantly, this protein serves as a complement-like opsonin and promotes phagocytosis of some Gram-negative bacteria in a mosquito hemocyte-like cell line. Chemical inactivation by methylamine and depletion by double-stranded RNA knockout demonstrate that this function is dependent on the internal thioester bond. This evidence of a complement-like function in a protostome animal adds substantially to the accumulating evidence of a common ancestry of immune defenses in insects and vertebrates.

Innate immune defense against malaria infection in the mosquito.

Anopheles gambiae, the most important vector of malaria, employs its innate immune system in the fight against Plasmodium. This can affect the propagative capacity of Plasmodium in the vector and, in some cases, leads to total refractoriness to the parasite. The components operating in the mosquito's innate immune system and their potential relevance to antimalarial responses are being systematically dissected.

Constitutive expression of a complement-like protein in toll and JAK gain-of-function mutants of Drosophila.

We show that Drosophila expresses four genes encoding proteins with significant similarities with the thiolester-containing proteins of the complement C3/alpha(2)-macroglobulin superfamily. The genes are transcribed at a low level during all stages of development, and their expression is markedly up-regulated after an immune challenge. For one of these genes, which is predominantly expressed in the larval fat body, we observe a constitutive expression in gain-of-function mutants of the Janus kinase (JAK) hop and a reduced inducibility in loss-of-function hop mutants. We also observe a constitutive expression in gain-of-function Toll mutants. We discuss the possible roles of these novel complement-like proteins in the Drosophila host defense.

Constitutive activation of toll-mediated antifungal defense in serpin-deficient Drosophila.

The antifungal defense of Drosophila is controlled by the spaetzle/Toll/cactus gene cassette. Here, a loss-of-function mutation in the gene encoding a blood serine protease inhibitor, Spn43Ac, was shown to lead to constitutive expression of the antifungal peptide drosomycin, and this effect was mediated by the spaetzle and Toll gene products. Spaetzle was cleaved by proteolytic enzymes to its active ligand form shortly after immune challenge, and cleaved Spaetzle was constitutively present in Spn43Ac-deficient flies. Hence, Spn43Ac negatively regulates the Toll signaling pathway, and Toll does not function as a pattern recognition receptor in the Drosophila host defense.

Two distinct pathways can control expression of the gene encoding the Drosophila antimicrobial peptide metchnikowin.

Metchnikowin is a recently discovered proline-rich peptide from Drosophila with antibacterial and antifungal properties. Like most other antimicrobial peptides from insects, its expression is immune-inducible. Here we present evidence that induction of metchnikowin gene expression can be mediated either by the TOLL pathway or by the imd gene product. We show that the gene remains inducible in Toll-deficient mutants, in which the antifungal response is blocked, as well as in imd mutants, which fail to mount an antibacterial response. However, in Toll-deficient;imd double mutants, metchnikowin gene expression can no longer be detected after immune challenge. Our results suggest that expression of this peptide with dual activity can be triggered by signals generated by either bacterial or fungal infection. Cloning of the metchnikowin gene revealed the presence in the 5' flanking region of several putative cis-regulatory motifs characterized in the promoters of insect immune genes: namely, Rel sites, GATA motifs, interferon consensus response elements and NF-IL6 response elements. Establishment of transgenic fly lines in which the GFP reporter gene was placed under the control of 1.5 kb of metchnikowin gene upstream sequences indicates that this fragment is able to confer full immune inducibility and tissue specificity of expression on the transgene.

Metchnikowin, a novel immune-inducible proline-rich peptide from Drosophila with antibacterial and antifungal properties.

One of the characteristics of the host defense of higher insects is the rapid and transient synthesis of a variety of potent antimicrobial peptides. To date, several distinct inducible antimicrobial peptides or peptide families have been totally or partially characterized. We present here the isolation and characterization of a novel 26-residue proline-rich immune-inducible peptide from Drosophila, which exhibits both antibacterial (Gram-positive) and antifungal activities. Peptide sequencing and cDNA cloning indicate the presense of two isoforms in our Drosophila Oregon strain, which differ by one residue (His compared to Arg) as a consequence of a single nucleotide change. The gene, which maps in position 52A1-2 on the right arm of the second chromosome, is expressed in the fat body after immune challenge. The novel peptide, which we propose to name metchnikowin, is a member of a family of proline-rich peptides, and we discuss the possible evolutionary relationships within this family.